SUMMARY: Antiretroviral therapy (ART) is the most effective means for reducing mortality and morbidity of individuals infected with HIV infection, however this therapeutic strategy is costly and is not tolerated well in many individuals. We are now challenged with the goal of functionally curing individuals that have ART suppressed HIV. The goal of this proposal is define the DNA methylation programs involved in limiting the survival and effector function of HIV-specific T cells and to optimize gene editing approaches to develop an HIV-specific T cell receptor (TCR) T-cell that can provide long-lived control of the virus in chronically infected individuals. To achieve this goal, we have developed a research program that is focused on understanding the mechanism for acquisition and maintenance of acquired exhaustion gene expression programs in TCR T cells. Notably, our group has recently reported that conditional deletion of the DNA methyltransferase 3a (Dnmt3a cKO) in virus-specific CD8 T-cells prevents functional T-cell exhaustion and preserves a long-lived population of T-cells that retain a heightened capacity to mount an antiviral response during a chronic viral infection. The following aims build upon this published work and will facilitate the design of a HIV-specific CAR T cell, as well as identify epigenetic biomarkers that can be used to assess CAR T cell exhaustion in future clinical trials. The specific aims of this proposal are: Aim 1. To determine if DNMT3a-mediated methylation of stemness-associated genes is coupled to the functional and clonal breadth of HIV-specific CD8 T cells during natural viral control. Aim 2. To determine if DNMT3a programming is coupled to reduced self-renewal and effector functions of endogenous and receptor-engineered HIV-specific T cells. Aim 3. To determine if de novo DNA methylation blockade enhances in vivo TCR T cell mediated HIV viral control and memory generation. Completion of the proposed studies will define the epigenetic programs that limit endogenous and engineered HIV-specific T-cell survival and effector functions, and will advance strategies for generating adoptiveT-cell therapy that provide durable HIV viral control.